TWI405566B - Use of curcumin or its analogues in cancer therapy utilizing epidermal growth factor receptor tyrosine kinase inhibitor - Google Patents

Abstract

Provided is combined use of an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and curcumin or its analogue in cancer therapy, which reduces side effects resulting from the EGFR-TKI and reduces doses of the EGFR-TKI needed for the therapy, particular in a patient resistant to the treatment with the EGFR-TKI alone.

Description

Use of curcumin or its analogs in cancer medicine using an epithelial cell growth factor receptor tyrosine kinase inhibitor

The present application claims priority to U.S. Provisional Patent Application Serial No. 61/303,593, filed on Jan.

The present invention relates to the use of curcumin or an analogue thereof for cancer therapy using an epithelial cell growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), which reduces the side effects caused by treatment and reduces the need for treatment. EGFR-TKI dose, especially in patients who are resistant to EGFR-TKI alone.

Cancer is the leading cause of death in the world. Recently, so-called "target therapy" has been developed, and drugs that selectively act on the epithelial growth factor receptor (EGFR) have been clinically proven ( Cancer Research 2004; 64(15): 5355-62). ). The signaling pathway of EGFR is one of the major factors regulating cell growth and differentiation, which regulates phosphorylation by endogenous tyrosine kinase ( Cancer Research 2003 ; 63(1): 1-5). Excessive expression of EGFR was found in different malignant cells with a poor prognosis ( Oncologist 2004; 9(1): 58-67). However, some key issues still limit the use of these drugs.

Iressa Orally effective EGFR-TKI, the first selective small molecule drug approved for the treatment of non-small cell lung cancer (NSCLC) ( Lung Cancer 2003; 41 Suppl 1: S9-14; and Expert Review of Anti Cancer Therapy 2004) ;4(1):5-17). Previous studies involving multi-agency clinical trials have confirmed that Asian patients respond better to Aressa than white patients and are best at women with non-smoking adenocarcinoma ( Proceedings of the National Academy of Sciences of the United States) Of America 2004; 101(36): 13306-11; and Lancet 2005; 366 (9496): 1527-37). Recent studies have indicated that EGFR exon 19 fragment deletion (ΔE746-A750) and exon 21 L858R substitution in NSCLC are highly correlated with Ares sensitivity ( New England Journal of Medicine 2004; 350(21): 2129-39; Science 2004; 304 (5676): 1497-500; and Oncologist 2008; 13(12): 1276-84). However, the EGFR gene mutation rate in NSCLC patients has been found to range from 10% to 15% in Caucasians and from 30% to 40% in Asians ( Clinical Cancer Research 2008; 14(10): 2895-9 ; and Journal of the National Cancer Institute 2005; 97 (5): 339-46). In all NSCLC cases worldwide, patients with wild-type EGFR still accounted for the majority, and these people tested a relatively poor response to Aretha treatment. Further, substitution by the second site, in addition to EGFR exon 20 T790M, caused by acquired resistance, resulting in poor Aretha activity (New England Journal of Medicine 2005; 352 (8): 786-92; And PLoS Medicine/Public Library of Science 2005; 2(3):e73).

Side effects are another limitation of using EGFR-TKI. It is known that Aressa causes side effects such as diarrhea ( Journal of Clinical Oncology 2003; 21(12): 2237-46; and Clinical Cancer Research 2004; 10(4): 1212-8) and rash. The frequency of diarrhea caused by Aretha in clinical trials was 67% in the 500 mg/day group and 48% in the 250 mg/day group. A recently published case has shown that the combination of EGFR-TKI and radiation therapy can lead to unintended toxicity and fatal diarrhea in patients with metastatic NSCLC ( Lung Cancer 2008; 61(2): 270-3). These side effects can cause physical and psychological discomfort, which can lead to dose reduction or treatment interruption.

The present invention is based on the discovery of a curcumin-based potential agent which reduces the side effects caused by EGFR-TKI treatment and reduces the dose of EGFR-TKI required for the treatment of EGFR-TKI cancer, particularly for EGFR-TKI alone. Resistant patient.

Accordingly, in one aspect, the invention provides a method for reducing side effects caused by treatment with an epithelial cell growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), comprising administering an effective amount that reduces such side effects Curcumin or an analog thereof to a patient receiving this treatment. In a specific embodiment, the side effects are gastrointestinal-induced side effects of EGFR-TKI, such as intestinal cell damage or growth inhibition.

In another aspect, the present invention provides a method of administering an epithelial cell growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) to a patient in need of cancer therapy using EGFR-TKI, comprising administering a reduced dose of EGFR-TKI. Curcumin or an analogue thereof to the patient, and the efficacy of the EGFR-TKI in cancer medical treatment is compared to the effect of administering only a standard dose of EGFR-TKI without administration of curcumin or its analogs. It is maintained. In a specific embodiment, the patient is diagnosed as having EGFR-TKI resistance. In a specific embodiment, the reduced dose is about 50% or less of the standard therapeutic dose of EGFR-TKI.

In a specific embodiment, the patient to be treated has non-small cell lung cancer (NSCLC). In another specific embodiment, the patient to be treated is EGFR-TKI resistant.

In a specific embodiment, the curcumin analog is selected from the group consisting of:

In a specific embodiment, the EGFR-TKI is Arisa (N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-propoxy) Quinazoline-4-amine).

In a specific embodiment, the curcumin or analog thereof is administered concurrently with EGFR-TKI.

The invention also provides a method for treating a cancer patient having EGFR-TKI resistance comprising co-administering an effective amount of EGFR-TKI and curcumin or an analog thereof to the aforementioned patient.

Various specific embodiments of the invention are detailed below. Other features of the present invention will be apparent from the following detailed description and drawings.

It is believed that those skilled in the art having the present invention can use the invention to the broadest scope thereof without departing from the scope of the invention. Accordingly, the following description is to be considered as illustrative and not restrictive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art. All publications mentioned herein are hereby incorporated by reference in their entirety to the extent of the disclosure of the disclosure of the disclosure of the disclosure.

As used herein, the singular forms "a", ""," Thus, for example, reference to "the same" includes a plurality of such samples and equivalents known to those skilled in the art.

As described above, the present inventors have found that administration of curcumin or an analog thereof to a patient undergoing EGFR-TKI treatment can reduce side effects caused by the treatment.

Accordingly, in one aspect, the invention provides a method for reducing side effects caused by treatment with an epithelial cell growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) comprising administering an effective amount that reduces such side effects Curcumin or an analog thereof to a patient receiving this treatment.

The epithelial cell growth factor receptor (EGFR) is a 170 kDa membrane-bound protein that is expressed on the surface of epithelial cells, which are known to be involved in the regulation of cell growth and differentiation, and are regulated by intrinsic tyrosine-induced phosphoric acid. Chemical. A large number of EGFR manifestations have been reported in various malignant cells and are associated with poor prognosis. As disclosed herein, the disclosed EGFR protein is GenBank Accession No. NM_005228 (SEQ ID NO: 1).

The term "EGFR-TKI" as used herein refers to an epithelial cell growth factor receptor tyrosine kinase inhibitor. Some examples of EGFR-TKI include Arisa, also known as N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-propoxy)quina Oxazolin-4-amine (Iressa ), as well as erlotinib, ie N-(3-acetylenephenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (Tarceva ), which is a clinically used drug for the treatment of non-small cell lung cancer.

The term "side effects" as used herein refers to side effects induced by EGFR-TKI, such as adverse gastrointestinal effects (eg, diarrhea, intestinal villus/cell damage, or growth inhibition of intestinal cells) or adverse skin conditions (eg, Rash or dry skin). In a specific embodiment, the side effect is EGFR-TKI-induced intestinal cell damage or growth inhibition.

Curcumin (diferuloylmethane) is a highly active ingredient extracted from the plant Curcuma longa . The chemical formula (enol type) is as follows:

The term "curcumin" as used herein also includes analogs, derivatives, or salts thereof. Products made from curcumin, such as food additives or supplements, are also included. In a specific embodiment, the curcumin analog is selected from the group consisting of LL-17, LL-18, LL-68, and LC-15, and the chemical formula is as follows:

The curcumin or an analog, derivative or salt thereof used herein may be according to a general method known in the art, for example, Bioorganic & Medicinal Chemistry 2006; 14(8): 2527-34; and Journal of Medicinal Chemistry 2006 ; 49(13): 3963-72, synthesized or isolated from natural resources.

The terms "patient", "individual" and "individual" as used herein are used interchangeably and specifically refer to a human subject in need of cancer care. In a specific embodiment, the individual has non-small cell lung cancer (NSCLC).

According to the invention, EGFR-TKI and curcumin or analogues thereof may be administered in a therapeutic dosage form, or in separate therapeutic doses, such as separate capsules, tablets, containers or injections. EGFR-TKI and curcumin or analogs thereof can be administered simultaneously (in parallel) or sequentially. In a specific embodiment, EGFR-TKI and curcumin or an analog thereof are administered in parallel.

To facilitate delivery, EGFR-TKI and curcumin according to the invention may be formulated, individually or in combination, into a pharmaceutical composition having a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient contained in the composition, preferably stabilizing the active ingredient, and is not deleterious to the subject being treated. The carrier can act as a diluent, carrier, excipient, or vehicle for the active ingredient. Examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, goat thorn, gelatin, calcium citrate, microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The pharmaceutical composition may additionally comprise a lubricant such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifier and a suspending agent; a preservative such as methyl paraben and propyl paraben; a sweetener; And flavoring agents.

The pharmaceutical composition according to the present invention may be in the form of tablets, pills, powders, lozenges, sachets, sachets, medicinal liquors, suspensions, emulsions, solutions, syrups, soft and hard capsules, suppositories, aseptic injection, and in packaged powder form. .

The pharmaceutical compositions of the present invention can be delivered via any physiologically acceptable route, such as oral, parenteral (e.g., muscle, vein, subcutaneous, abdominal), transdermal, rectal, inhalation, and the like. In a specific embodiment, the compositions of the invention are administered orally.

In the present invention, it has also been found that when EGFR-TKI is administered in combination with curcumin to a patient in need thereof, the effective amount of EGFR-TKI can be reduced, and the medical effectiveness thereof is substantially the same as that of EGFR-TKI alone. It is maintained.

Accordingly, in another aspect, the present invention provides a method of administering an epithelial cell growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) to a patient in need of cancer therapy using EGFR-TKI, comprising administering a reduced dose of EGFR- TKI combines curcumin or an analog thereof into the patient, and the efficacy of the EGFR-TKI in cancer medical treatment is comparable to that of standard dose EGFR-TKI administered without the administration of curcumin or its analogs. , is essentially maintained.

An "effective amount" or an "effective amount" for administering a pharmaceutical agent means an amount or dose which results in a desired pharmaceutical result, such as improving symptoms, reducing side effects, prolonging life or improving quality of life; Individually, for example, the rate of tumor growth is reduced, the volume of the tumor is reduced, or the tumor is completely eliminated. The effective amount or dosage of a medicament may vary depending on the particular active ingredient employed, the mode of administration, the age, weight and condition of the individual being treated. The exact amount of administration of the desired agent is in accordance with the judgment of the medical practitioner and is unique to each individual.

As used herein, the term "standard dose" refers to an effective dose of a therapeutic agent that is an authority in the pharmaceutical industry, including the Food and Drug Administration, and is often used for routine practice. As used herein, the term "reduced dose" refers to a dose that is lower than the standard dose but still substantially retains the same therapeutic effect of the same therapeutic agent. In particular, according to the present invention, the reduced dose of EGFR-TKI is about 90% or less, 80% or less, 70% or less, 60% or less, 50% of the standard therapeutic dose of EGFR-TKI. Or less. In a particular embodiment of the invention, the reduced dose is about 50% or less of the standard therapeutic dose of EGFR-TKI.

It is known that some patients have a poor response to EGFR-TKI therapy and may require high doses to achieve the desired therapeutic effect, however, this can result in unacceptable toxicity to the patient and ultimately lead to treatment discontinuation. Surprisingly, the method of the invention is particularly effective in treating patients with EGFR-TKI resistance. When administered to a patient who is resistant to EGFR-TKI, the method of the invention allows administration of a reduced dose of EGFR-TKI to the patient, and its medical effectiveness is substantially compared to the administration of EGFR-TKI alone. maintain. In a specific embodiment, the patient has a mutation that is resistant to EGFR-TKI, for example, a T790M substitution in EGFR (SEQ ID NO: 1).

The invention also provides a method for treating a cancer patient having EGFR-TKI resistance comprising co-administering an effective amount of EGFR-TKI and curcumin or an analog thereof to the aforementioned patient.

As used herein, the term "treatment" refers to the use or administration of a composition comprising one or more active agents to a subject, in particular, the individual having a condition to be treated with EGFR-TKI, including but not limited to, A tumor or cancer, a symptom of a disease, or a predisposition to a disease, which cures, heals, alleviates, alleviates, alters, remedies, soothes, improves, or affects the disease, the symptoms of the disease, or the predisposition to the disease. purpose.

The present invention will now be described more specifically with reference to the following specific embodiments, which

Materials and Methods

Reagent

Curcumin (purified by HPLC: 98.0%) for in vitro studies was purchased from Calbiochem (Darmstadt, Germany). In vivo, curcumin (purity ~70%) is from Sigma (St Louis, MO, USA). Aretha ( ), ZD1839, provided in good faith by Astra-Zeneca Pharmaceuticals (Macclesfield, UK). Curcumin and Arisha stock solutions were prepared in dimethyl hydrazine (DMSO) and stored at -20 °C. For each experiment, the compounds were diluted in fresh media and the final concentration of DMSO was less than 0.1%. Curcumin and Elisa for animals are prepared by completely suspending the drug in propylene glycol (JT Baker, Phillipsburg USA).

2. Cell lines and culture conditions

A highly invasive human lung adenocarcinoma cell line (CL1-5) has been established ( American Journal of Respiratory Cell & Molecular Biology 1997; 17(3): 353-60). Human lung cancer cell lines A549, H1299, H1650, and H1975 were obtained from the American Type Culture Collection (Manassas, VA). The PC-9 is a gift from Dr. Chih-Hsin Yang (Taiwan University Hospital, Taiwan). These cells were grown in RPMI 1640 medium (Life Technologies Rockville, MD). The IEC-18 rat intestinal epithelial cell line (BCRC 60230) was grown in DMEM medium (Life Technologies Rockville, MD) supplemented with 10% fetal bovine serum (FBS) (Life Technologies). The above medium contained penicillin and streptomycin (100 mg/ml each) and the cell line was cultured at 37 ° C in a humidified environment with 5% CO ₂ .

3. Proliferation test

MTT [3-(4,5-dimethylthiazolyl-2)-2,5-brominated diphenyltetrazolium] (Sigma, St Louis, MO) assay was performed to determine the measurement of cell proliferation. Briefly, CL1-5, A549, PC-9, H1650, H1975, and IEC-18 cells were plated in 96-well plates at a density of 5 x 10 ³ cells/well. After 24 hours of culture, cells were treated with different concentrations of curcumin and/or Aesar for 72 hours. In addition, IEC-18 cells were treated with curcumin, BIRB 796 and/or Aretha for 24 hours. The final concentration of the MTT solution in the medium was 0.5 mg/ml and was added to the wells. After a further 1.5 hours of incubation, the medium was removed and DMSO was added to the dish. The color rendering intensity of the dissolved formazan was measured at 570 nm using a multi-label plate reader (Vector3; Perkin-Elmer, USA).

4. Colony formation test

CL1-5, A549 and H1975 cells were plated in 6-well plates with medium (100 cells per well). After 24 hours of incubation, the cells were treated with either Aressa or Curcumin alone or in combination as indicated. The cells were cultured with the reagents for 5 days, followed by complete replacement of the medium; and the cells were further cultured for 9 days. Colonies were stained with 0.001% crystal violet and the number of colonies per well was calculated.

5. Western blot analysis

Western blotting method was used to determine the protein expression of EGFR, pEGFR, Akt, pAkt, cyclin D1, PCNA, iNOS and various apoptosis-related proteins (caspase-3, 8, 9 and PARP). . The cells were plated on a 10-cm dish with a density of 1 x 10 ⁶ . After culturing overnight, cells were treated with serum starvation for 24 hours in FBS-free medium. Cells were then treated with different concentrations of curcumin and/or Aesar for 1 hour under serum free conditions, followed by stimulation with 20 ng/mL EGF for 30 minutes. IEC-18 cells were plated in 10-cm dishes at a density of 1 x 10 ⁶ . After overnight incubation, cells were treated with different concentrations of curcumin and/or Aretha or BIRB 796 for another 24 hours. The cells were washed three times with ice-cold PBS and their proteins were extracted. In addition, CL1-5 tumor tissue (100 mg) was obtained from each group and then minced in lysis buffer. Protein extracts were obtained using a mammalian protein extraction reagent (Pierce, Rockford) containing a protease inhibitor and a phosphatase inhibitor (Sigma, USA). SDS/PAGE was performed using 10% analytical gel to separate proteins (25 mg/adhesive). Anti-phospho-EGFR (Tyr1068), phospho-Akt (Ser473), Akt, c-MET, caspase-3, caspase-8, caspase-9, PARP, active-p38 The anti-system of p38 was purchased from Cell Signaling Technology (Beverly, MA). Anti-EGFR, cyclin D1, PCNA, and iNOS forms of the anti-system were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). These anti-systems are used according to the manufacturer's recommendations. Linked antibodies were detected using an enhanced chemical luminescence system (Santa Cruz, CA). Chemical luminescence signals were captured using a Fujifilm LAS 3000 system (Fujifilm, Tokyo, Japan). All experiments were repeated three times.

6. Flow cytometry

The seed cells were seeded in a 6-mm culture dish at a density of 10 ⁵ cells per dish. After 24 hours of culture, the cells were treated overnight by serum starvation. The cells were then treated with curcumin and/or Ares for another 72 hours. Adherent and floating cells were separately collected and resuspended in cold 1x PBS for further analysis. Cells were stained with annexin V-FITC cell apoptosis kit (BD Pharmingen, USA) to monitor apoptotic cells and stained with propidium iodide (PI) to detect dead cells. Samples were analyzed in a FC 500 flow cytometry system (Beckman Coulter). Unstained cells were classified as "alive", and cells infected with annexin V alone were classified as "early apoptosis", and cells infected with both annexin V and PI were classified as "late apoptosis". Cells that are only infected with PI are classified as "death."

7. Real-time quantitative RT-PCR

The amount of EGFR expression was detected by real-time PCR on an ABI prism 7900 Sequence Detection System (Applied Biosystems). The EGFR primers used were as follows: pre-introduction EGFR-F, 5'-GTGACCGTTTGGGAGTTGATGA-3'; inverted primer EGFR-R, 5'-GGCTGAGGGAGGCGTTCTC-3'. TATA-box binding protein (TBP) was used as internal control (GenBank X54993). The primers and probes for quantitative RT-PCR of TBP mRNA were as previously described (24, 25). The relative performance of EGFR compared to TBP is defined as -ΔCT=-[CT _EGFR -CT _TBP ]. The EGFR mRNA/TBP mRNA ratio was calculated as 2 - ^ΔCT × K, where K is a constant. All experiments were repeated three times.

8. In vivo research protocol

We conducted in vivo studies in mice according to the experimental protocol approved by the National Yang Ming University Animal Care and Use Committee. CL1-5 cells were calculated using trypan blue according to cell survival and cell number, followed by subcutaneous injection of 1×10 ⁶ live CL1-5 cells into 6-week large SCID mice in 100 μl HBSS (by the Animal Center of Taiwan University Medical School, Taipei, Taiwan, provided). In order to test whether curcumin can enhance the anti-tumor effect of Aressa, mice were arbitrarily divided into five groups (n=9) one week after cell injection; these were: (1) empty vector control group; (2) curcumin alone Group (1 g/kg); (3) Aressa alone group (120 mg/kg); (4) 60 mg/kg Aressa plus 1 g/kg curcumin group; and (5) 120 mg/kg Aressa plus 1 g/kg curcumin group. The curcumin and esarea dosed as indicated are administered orally to the animals daily. The tumor volume was visualized every four days using an electronic vernier caliper and the tumor volume was calculated using the formula V = 0.4 x ab ² , where a and b are the longest and shortest diameters of the tumor, respectively. After 3 weeks, the mice were sacrificed and the subcutaneous tumor was excised; then frozen in liquid nitrogen and finally stored at -80 °C. Intestinal samples from each group of mice were fixed and paraffin embedded using Bouin's fluid for routine hematoxylin and eosin staining.

9. PCNA immunohistochemical staining, and cell apoptosis detection test

Cell proliferation assays were performed on paraffin-embedded tumor tissue samples using PCNA staining. Briefly, a rabbit anti-human PCNA polyclonal antibody (Santa Cruz, CA) was used in one reaction. The DAKO EnVision system was used with 3,3'-diamine benzidine containing a secondary horseradish peroxidase-conjugated anti-mouse antibody complex to detect PCNA.

Colorimetric immunohistochemical staining (TUNEL) for apoptotic cell death was performed on paraffin-embedded tumors and intestinal tissue sections using an in situ cell death detection kit, POD (Roche Diagnostics, Germany); Gill's hematoxylin contrast stained the sections. TUNEL-positive cells were tested in 10 random fields from each of the three intestinal tracts of each treatment group, followed by a statement of the mean number of TUNEL-positive cells ± SE per high power field (x400 magnification).

10. Measurement of caspase activity

By using caspase-Glo The 3/7 test kit (Promega Corporation, Australia) detected caspase activity. Briefly, seed crystal IEC-18 cells were seeded at a density of 1 x 10 ⁴ cells per well in a 96-well white photometer test dish and cultured at 37 °C. After 24 hours of culture, cells were treated with different concentrations of Ares and/or curcumin for another 24 hours. 100 μl of caspase 3/7 reagent was added to each well and incubated for 1 hour at room temperature on a rotary shaker. The luminescence intensity of each well was measured using a multi-label reader (Vector3; Perkin-Elmer, USA).

11. Statistical analysis

All experiments were repeated three times and analyzed by ANOVA (Excel, Microsoft; Taipei, Taiwan). Comparisons were made using a two-tailed Stuart t-test and significant differences were defined as p < 0.05. Where appropriate, the data is expressed as mean + standard deviation (mean ± SD).

result

1. Curcumin can inhibit the proliferation of lung adenocarcinoma cells, EGFR and AKT protein expression and phosphorylation

In order to develop new agents or compounds that enhance anticancer effects, reduce doses, or overcome the resistance of NSCLC patients to Aretha, use high-throughput drug screening systems with different Aresar-resistant cell lines, Hundreds of compounds were screened from the herbs in our laboratory. Table 1 shows the EGFR status and race of the NSCLC cell line used herein.

Curcumin was chosen as a potential candidate. We confirmed that in the Nethera-resistant NSCLC cell line, including CL1-5 (wt-EGFR), A549 (wt-EGFR), H1299 (wt-EGFR), H1650 ( EGFR exon 19 fragment deletion (ΔE746-A750), but no PTEN protein), and H1975 (L858R and T790M mutations of EGFR), curcumin exhibited a significant inhibitory effect on cell proliferation. As shown in Figure 1A, after incubation with Ares for 72 hours, CL1-5, A549, H1299, H1650, and H1975 were shown as compared to the cell line PC-9 sensitive to Aressa (IC50 < 0.1 μM). The overall layout of the increased resistance (IC50 > 10 μM) (Fig. 1A); however, the proliferation of these Ariza resistant cells was inhibited by curcumin in a concentration-dependent manner (Fig. 1 right).

It is known that the EGFR message transmission pathway is highly correlated with tumor development, and therefore the effects of curcumin on the expression and activity (phosphorylation) of EGFR and AKT in CL1-5, A549 and H1975 cells were tested (Fig. 1B). The results indicated that curcumin reduced the expression of EGFR, pEGFR, AKT, and pAKT proteins in a concentration-dependent manner in both CL1-5 and A549 cells (Fig. 1B). Although curcumin did not alter AKT phosphorylation in H1975 cells, curcumin significantly reduced the amount of EGFR downstream signaling factor, cyclin D1 (Fig. 1B, right). Furthermore, RTQ-PCR results showed that curcumin reduced EGFR mRNA expression in a concentration-dependent manner (Fig. 1C). These results indicate that the curcumin dose exhibits a potential anticancer effect by attenuating EGFR signaling in the Aretha-resistant NSCLC cells.

2. The binding activity of curcumin to EGFR

We studied the binding activity of curcumin to EGFR and compared it with Aretha. The results showed that according to LIBDOCK, the predicted 3-D conformation of curcumin bound to the open-type wild-type EGFR protein had a relatively high score (curcumin score = 89.5; Aressa score = 80.3). Furthermore, Figure 2 shows that curcumin binds to EGFR tyrosine phosphorylation with a T790M resistance mutation. Taken together, these results show that curcumin exhibits a higher binding activity to EGFR than that of Aressa, and that the T790M resistance mutation does not affect the binding activity of curcumin to EGFR.

3. Curcumin promotes EGFR degradation and down-regulates EGFR protein content

We tested whether curcumin promotes degradation of EGFR during the translation phase. Lung adenocarcinoma cells were pretreated with MG132 for 3 hours or without pretreatment, followed by treatment with curcumin as indicated. Our data show that curcumin inhibits EGFR protein expression in a dose-dependent manner; however, when the cells are pretreated with MG132, which is a proteasome inhibitor, the EGFR protein concentration can be recovered (Fig. 3A). These results indicate that curcumin can reduce the amount of EGFR protein expression by promoting ubiquitin-proteasome capacity.

We also found that the concentration of EGFR protein in the Aressa combined curcumin group was lower than that in the curcumin alone group. Therefore, we performed an immunoprecipitation test to test this observation. The results showed that Aressa (10 μM) did not change the amount of EGFR ubiquitin. However, in the combined treatment group, Aressa (1 μM) and curcumin (10 μM) increased EGFR protein compared to the other treatment groups. Ubiquitination (Figure 3B). This data indicates that curcumin enhances the anti-tumor properties of Ares by down-regulating EGFR protein concentrations.

4. Combination of curcumin and aresal can improve the anti-tumor effect of Aressa in NSCLC cells, with wild type or mutation EGFR Everyone

Use of wild-type or mutant EGFR CL1-5, A549, H1299, H1650 and H1975 cell lines to assess whether curcumin can increase the anti-tumor effect of a variety of EGFR- containing Aressa-resistant NSCLC cells effect. Cell proliferation tests show that Aretha ( 10 μM) or curcumin ( 15 μM) treatment alone produced only a slight inhibition of cell proliferation (Fig. 4A). However, when combined with curcumin (15 μM) and Aressa (1 μM), cell proliferation was significantly reduced in five Nerve-resistant NSCLC cell lines, and the anti-proliferative effect was equivalent to High dose Aressa (20 μM) (Figure 4A). When the expression levels of EGFR, pEGFR, AKT and pAKT in CL1-5, A549 and H1299 cells were examined, it was also found that the combination of curcumin and Aisal significantly inhibited EGFR signaling. These results indicate that the two groups of Aressa (1 μM) or curcumin (10 and 15 μM) have only minimal inhibitory effects on pEGFR and pAKT (Fig. 4B), and conversely, combined with Aesar (1 μM) and curcumin (15 μM) produced a significant barrier to EGFR and AKT phosphorylation (Fig. 4B). These results suggest that curcumin enhances the anti-cancer effect of Aretha when using Aretha to treat Aretha-resistant cancer cells.

In addition, we investigated whether curcumin can increase the amount of apoptosis caused by Aretha in the CL1-5, A549, and H1975 cell lines. A cytometry assay using propidium iodide/Annexin-V staining indicated that in CL1-5 and A549 cells, the combination of Aressa (1 μM) and curcumin (15 μM) induced a higher concentration of Ai A higher amount of apoptosis was observed in the Resa alone group (20 μM) (Fig. 5A, left and middle). In H1975 cells, curcumin and Aressa also increased the number of annexin V-positive cells compared to the Aressa alone group, but this amount was similar to curcumin (15 μM) alone; 15 μM turmeric Whether or not there is Eriza, it can induce apoptosis of about 90% of H1975 cells (Fig. 5A, right), which explains that the two agents are not induced together. Taken together, these results seem to indicate that curcumin enhances apoptosis induced by Aretha in Elisa-resistant cells.

Next, we used a colony formation assay to test whether curcumin enhanced the anti-tumorigenicity of Aretha in CL1-5, A549 and H1975 cells. The results were similar to the MTT assay, and it was found that the combination of curcumin and Aesar significantly inhibited the colony formation of CL1-5, A549 and H1975 compared to the drug-free control group and the respective treatment groups (Fig. 5B). The above in vitro studies support the use of curcumin to enhance the anticancer activity of Aretha when using Aretha-treated resistant lung adenocarcinoma cells, a hypothesis; this effect does not appear to be mutated by EGFR mutants or Any other genetic variation.

5. Allogeneic transplantation in vivo, curcumin enhances the anti-tumor properties of Aretha in human lung adenocarcinoma cells.

The next step is to investigate whether curcumin can enhance the anti-tumor activity of Aressa in vivo. To this end, CL1-5 cells were implanted subcutaneously into SCID mice. One week later, when tumors were detectable (3-5 mm), mice were randomly divided into five groups (Fig. 6A). The five groups were then subjected to different treatment systems: empty vehicle as control group, curcumin (1 g/kg) group, Aressa (120 mg/kg) group, curcumin (1 g/kg) A combination of low-dose escar (60 mg/kg) and curcumin (1 g/kg) combined with high-dose essa (120 mg/kg). These treatments were given orally once a day and the mice were sacrificed four weeks later. During this time, the tumor volume of each group was monitored every four days (Fig. 6B). At the end of the study, the tumor size of the control group animals averaged 638.55 mm ³ . Significantly, the tumor size of the curcumin plus high-dose Ayerson combined treatment group showed the largest reduction to only 64.15 mm ³ ( p = 0.0001 control control group), whereas curcumin plus low-dose Ayresa group only The display was reduced to an average of 160.59 mm ³ ( p = 0.0006 control group). The mean tumor size of the curcumin alone group and the Arisha group were 354.91 ( p = 0.015 control group) and 138.32 mm ³ ( p = 0.0003 control group). Therefore, curcumin combined with the anti-tumor activity of Aressa showed an enhanced effect compared to the curcumin and Ayres alone groups ( p < 0.01 control curcumin group, and p = 0.015 control Aressa group). In addition, the group treated with low dose curcumin (60 mg/kg) showed similar anti-tumor activity ( p = 0.483) to the 120 mg/kg irissa alone group (Fig. 6C). Therefore, the combination of curcumin and low dose of Aressa can save half of the amount of Aressa and achieve the same inhibition of tumor progression. These results clearly show that curcumin enhances the anti-tumor activity of Aretha.

6. Curcumin enhances the anti-tumor effect of Aressa in vivo by reducing EGFR-related signaling and affecting the regulation of apoptosis.

In order to study the molecular mechanism involved in the anti-tumor activity of curcumin and Arisa combined mice, Western blotting analysis of protein lysates from various tumor tissues; this method is used to measure EGFR, AKT, cell cycle in tumors. The amount of protein D1, c-MET, PCNA, and iNOS protein. Figure 7A shows that the amount of EGFR and AKT in tumors of SCID mice was reduced after treatment with curcumin alone or curcumin and esa. In addition, the amount of protein of cyclin D1, c-MET, PCNA, and iNOS was significantly altered in these tumors (Fig. 7A, left). In order to confirm the apoptosis-inducing effect of curcumin in combination with Aesar in vivo, apoptosis-related signaling, including caspase and PARP, was studied. The results showed that curcumin enhanced the activity of caspase-3, caspase-8, caspase-9 and PARP, especially curcumin plus the Aressa group (Fig. 7A, right). These results indicate that curcumin appears to potentiate the anticancer activity of Aretha in cells that are resistant in vivo by reducing EGFR signaling, c-MET, PCNA, and iNOS, and by upregulating the apoptotic pathway.

We then examined the proliferating marker PCNA using immunohistochemical staining and assayed for cell death by the TUNEL assay; this was done in paraffin-embedded tissue samples. The results in Figure 7B show that PCNA labeling (Figure 7A, left) was reduced in the curcumin treated group and the curcumin combined with the Aressa group, which is similar to PCNA protein performance. In addition, the in situ cell death assay indicated that curcumin combined with the Aressa treatment group significantly increased the apoptotic activity of the cells compared to the control group and the Ares alone group (Fig. 7B). These results also showed that the curcumin alone group induced apoptosis of the cells compared to the control group (Fig. 7A, right). These results confirmed the anti-proliferative and apoptosis-inducing ability of curcumin in vivo.

7. Curcumin alleviates the adverse gastrointestinal effects of Aressa

During the study of in vivo allografts, the Aressa treated group showed striking weight loss (data not shown) and also had significant diarrhea side effects; this is similar to previous reports in the clinical literature, and even severe cases within the group death. Interestingly, when combined with arutha and curcumin, weight loss was avoided and the number of deaths between mice was significantly reduced (the survival rate of combined therapy was 78% compared to 33% of Aretha's therapy) ( Figure 8A, left). Next, the morphology and tissue of the intestine were examined to investigate the effect of curcumin on reducing the side effects of the gastrointestinal side of Aretha. The results showed that the length of the intestine of the Aressa group was shorter and thinner than that of the combination therapy group (Fig. 8A, right). There was no significant difference between the control group and the curcumin group, or between the curcumin and the Arisha group. Furthermore, when examined by H&E staining, the length of the intestine villi obtained from the combined group of curcumin and irissa was longer and had greater integrity than the Aressa treatment group ( p = 0.0001) (Fig. 8B). Furthermore, when evaluated in vivo by the TUNEL assay, curcumin combined with esarea significantly reduced apoptosis in the villus ( p = 0.0015) compared to the Aressa alone group (Fig. 8C). Therefore, curcumin can improve the survival rate of mice treated with Aretha, and can also reduce the side effects of GI of Aressa.

Finally, we investigated the protective effect of in vitro curcumin on apoptosis in the intestinal epithelial cells induced by Aretha, using the untransformed epithelial cell line IEC-18. Caspase-Glo 3/7 trials showed that Arisa (in terms of IEC-18 cells with IC ₂₅ at 30 μM and IC ₅₀ at 40 μM) could induce caspase 3/7 activity in IEC-18 cells. However, 5 μM curcumin (non-toxic dose) significantly inhibited the Aresar-induced caspase 3/7 activity (Fig. 9A). The results showed that curcumin prevented intestinal epithelial cells from being induced by Aressa; this effect was significantly similar to previous observations in vivo. We then determined which possible mechanisms involved the protective effect of curcumin on the apoptosis induced by Aretha. Previous reports have indicated that Aressa can induce apoptosis in intestinal epithelial cells via activation of protein kinases (MAPK), which is dependent on p38 mitogens ( Gatrointestinal & Liver Physiology 2007;293(3):G599- 606) This is a possible mechanism for the side effects of Aretha-related GI. Here, we found that active-p38 increased significantly with dose-dependent (0-20 μM) in IEC-18 cells, but CL1-5 cells did not increase p38 activation under the same culture conditions. (Fig. 9B). In addition, the Elisa-induced p38 MAPK activation in IEC-18 cells was significantly inhibited by curcumin: the results indicated that curcumin (5 μM) and the selective p38 MAPK inhibitor BIRB 796 (10 nM) were significantly Decrease the activity-p38 performance of Aretha-treated IEC-18 cells (Fig. 9C, left). Furthermore, MTT assays showed that Aressa (30 and 40 μM) significantly reduced IEC-18 cell viability, however, curcumin (5 μM), and BIRB 796 (10 nM) significantly significantly removed cells from The toxic effect of Aretha was rescued (Fig. 9C, right). Curcumin analogs, LL-17, LL-18, LL68, and JC-15, were also tested and found to have a protective effect against Areher-induced intestinal cell death (Fig. 10). Taken together; these results show that curcumin can reduce the complications induced by Aretha in the intestine.

In conclusion, our results show that curcumin acts as an outstanding activity of the anti-tumor enhancer of Aressa. This agent also alleviates the side effects of diarrhea in Aressa and is therefore a good adjuvant for patients with lung cancer. Clinically, the cost of lung cancer target therapy is quite high, and side effects are often a critical issue during treatment. Curcumin is a popular and less expensive agent, and it appears to enhance the effects of Aretha and thus reduce medical costs and patient financial burden. In order to reduce the dose of Aressa, cut costs, and avoid side effects, we recommend that curcumin should be a good adjuvant for patients with NSCLC during the treatment of Aretha.

For the purpose of illustrating the invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited by the preferred embodiments shown.

In the schema:

Figure 1 shows that curcumin potentially inhibits cell proliferation and reduces ligand-induced EGFR signaling activation in Nethera-resistant NSCLC cells. Part A, left, MTT test results showed that all six lung adenocarcinoma cell lines were susceptible to Aressa; right, MTT assay showed turmeric in five Nerve resistant NSCLC cell lines All of them cause inhibition of cell proliferation in a dose-dependent manner. Column, mean (n=6); strip, standard deviation (SD). The data system represents two separate experiments. In part B, Western blotting showed that curcumin dose-dependently (1-20 μM) reduced EGF (20 ng/ml)-induced EGFR expression, pEGFR concentration, AKT in CL1-5, A549 and H1975 cell lines. Performance, pAKT concentration, and cyclin D1 performance. The data series represents two independent experiments using β-actin as an internal control. Part C, real-time quantitative RT-PCR showed that in CL1-5 cells, EGFR mRNA expression was reduced by curcumin concentration-dependent (1-20 μM) after 24 hours of culture. Column, average (n=3); strip, SD. *, p < 0.05, which is statistically significant compared to the empty vehicle-treated control. The data system represents three separate experiments.

Figure 2 shows the binding of curcumin to the tyrosine phosphorylation site of EGFR with a T790M resistance mutation.

Figure 3 shows that curcumin accelerates the degradation of EGFR and down-regulates the protein concentration of EGFR. In part A, Western blotting showed that curcumin inhibited EGFR protein expression in a dose-dependent manner, whereas when the cell line was pretreated with MG132, the EGFR protein concentration was restored. In part B, immunoprecipitation experiments showed that essa (1 μM) and curcumin (10 μM) increased ubiquitination of EGFR protein compared to other single treatment groups.

Figure 4 shows that in vitro use of curcumin enhances anti-cell proliferation caused by Aretha and blocks EGFR signaling activation by Aretha. In section A, MTT assay showed that curcumin enhanced the anti-proliferative effect of Aretha in five lung cancer cell lines that were resistant to Aretha. Column, average (n=6); strip, SD. **, p <0.01. The data system represents two separate experiments. In part B, Western blotting showed that curcumin increased EGF (20 ng/ml)-induced EGFR and further reduced pEGFR, AKT and pAKT proteins after treatment of CL1-5, A549 and H1299 NSCLC cell lines with Aressa Performance concentration. The data series represents two independent experiments using β-actin as an internal control.

Figure 5 shows that in vitro NSCLC cells, curcumin enhances the apoptosis effect of Arisa and the inhibition of colony formation. In part A, Annexin V-FITC cell apoptosis assay showed that curcumin enhanced the apoptosis effect of Aressa; in the upper part, CL1-5 cells were treated with the indicated agents. In all the presented graphs, the x- axis is annexin-V-FITC, the y- axis is PI (propidium iodide); the lower part is calculated for CL1-5, A549 and H1975 cells undergoing apoptosis; Concentrations of annexin-V+ and PI- (early apoptosis) and annexin-V+ and PI+ (late apoptosis) in total cells were found in the lower right and upper right quadrants. Column, average (n=3); strip, SD. *, p < 0.05, **, p < 0.01 and p = 0.331 (15 μM curcumin versus 1 μM Aressa plus 15 μM curcumin). The data series represents independent experiments that have been done three times each. In part B , colony formation assays showed that curcumin enhanced the colony inhibition ability of Aretha in CL1-5, A549 and H1975 lung adenocarcinoma cells. Indications for treatment with cells; V: empty vector control group, G1, G5, G10, G15, C1, C5, C10, C15, G1+C1, G1+C5, G1+C10 and G1+C15: G and C lines Refers to Aretha and Curcumin, as well as the concentration of the digital display agent (μM). Colonies were counted after two weeks. Column, average (n=3); strip, SD. The data system represents two separate experiments.

Figure 6 shows that curcumin enhances the anti-tumor activity of Aressa in vivo. In part A, the in vivo experimental protocol is presented in a schedule. Part B, 10 ⁶ CL1-5 lung adenocarcinoma cells were implanted subcutaneously in SCID mice and the tumor volumes of the five groups were monitored over time. Point, the average of at least six mice per group; strip, SD. Part C, top panel, subcutaneous tumor photography cut from mice, indicates that curcumin enhances the anti-tumor growth activity of Aressa compared to the treated group alone; the following table shows the tumor volume on the last day of the experiment. Column , average (n=6); strip , SD. p = 0.0003 (control group control 120 mg/kg Aressa alone group), p = 0.0006 (control group control 60 mg/kg Aressa and 1 g/kg combination group), and p=0.484 (120 mg/kg) Aressa alone controls 60 mg/kg Aressa and 1 g/kg combined group).

Figure 7 shows the enhancement of anti-tumor proliferative activity and apoptosis induced by treatment with a combination of Iressa/curcumin using a xenograft tumor model. Part A, left, Western blotting shows that curcumin tuberculosis rossa inhibits the expression of EGFR, AKT, c-MET, cyclin D1, PCNA, and iNOS in lung adenocarcinoma tumor tissues; right, western blotting The method shows that curcumin combined with Aesar inhibits the expression of caspase-8, caspase-3 and caspase-9, and enhances the full length of PARP fragments in lung adenocarcinoma tumor tissues. The amount. The data is displayed using β-actin as an internal control. Samples were pooled from three animals in each group and analyzed; representative results were shown. Part B, H&E staining, proliferation-labeled PCNA, and immunohistochemical staining for apoptosis detection using the TUNEL assay. To this end, subcutaneous tumor sections of each group were used, and the results indicated that curcumin further inhibited tumor cell proliferation and enhanced apoptotic cell death induced by Aretha.

Figure 8 shows that curcumin reduces the gastrointestinal side effects of Aressa in vivo. Part A, left, the survival rate of each group of animals indicated that curcumin reduced the death of mice caused by Aretha's side effects; right, the photography of the intestines of each group of mice, presented to show that curcumin reduced the amount of Aressa Gastrointestinal side effects. Part B, left, H&E staining of the intestinal sections of each group showed that curcumin could prevent villus damage, which is one of the main side effects of Aretha; right, quantify the length of the villi (bar = 200 μm). Column, average (n=3); strip, SE. p = 0.0001 (120 mg/kg espressa alone group control 120 mg/kg irissa plus 1 g/kg curcumin group). Part C, left, use the gut sections of each group to detect apoptosis by TUNEL assay; these indicate that curcumin attenuates cell death caused by Aretha in the villi; right, TUNEL by the material and method described Quantification of staining positive cells. Column, average (n=10); strip, SE.

Figure 9 shows that curcumin reduces the gastrointestinal side effects of in vitro espressa. Part A, Caspase-Glo The results of 3/7 test showed that in IEC-18 cells, escarcin induced a dose-dependent induction of caspase 3/7 activity, but curcumin reversed this arisa-induced caspase. active. Column, average (n=3); strip, SD. The data system represents two separate experiments. In part B, Western blotting showed that Arisa dose-dependently (0-20 μM) increased the activity of p38, but the dose concentration did not affect CL1-5 cells. The data series represents two independent experiments using β-actin as an internal control. Part C, left, Western blotting shows that in IEC-18, curcumin and BIRB 796 reduced the concentration of p38 active protein induced by Aretha. The data series represent two independent experiments using β-actin as internal control; right, MTT assay showed that curcumin and BIRB 796 prevented IEC-18 cell proliferation from being erosive-induced toxic damage. Column, average (n=6); strip, SD. The data system represents two separate experiments.

Figure 10 shows that curcumin and its analogs LL-17, LL-18, LL-68, and JC-15 (columns 3-7) can alleviate the inhibitory effects of ERS-18 induced intestinal cell proliferation. Column, average (n=6); strip, SD. *, p < 0.05 and ** p < 0.01 compared to the Aressa treatment group (column 2). The data system represents two separate experiments.

Figure 11 shows the amino acid sequence of the EFGR protein, Accession No. NM_005228 (SEQ ID NO: 1) of GenBank.

<110> National Taiwan University

<120> Use of curcumin or an analogue thereof for cancer medical treatment using an epithelial cell growth factor receptor tyrosine kinase inhibitor

<130> IN0071/NTU0013US

<150> US61/303,593

<151> 2010-02-11

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1210

<212> PRT

<213> Human

<400> 1

Claims (7)

A use of curcumin or an analogue thereof for the manufacture of a medicament for reducing side effects caused by treatment with an epithelial growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), wherein the analogue is selected from A group consisting of: The use of the first aspect of the patent application, wherein the side effects are gastrointestinal-induced side effects of EGFR-TKI. The use of the second aspect of the patent application, wherein the side effects are EGFR-TKI-induced intestinal cell damage or growth inhibition. The use of the first aspect of the patent application, wherein the EGFR-TKI is Arisa (N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4- Propoxy)quinazolin-4-amine). For example, the application of the scope of claim 1 wherein the patient suffers from non-small Cell lung cancer (NSCLC). The use of the first aspect of the patent application, wherein the drug is administered simultaneously with EGFR-TKI. The use of any one of claims 1-6, wherein the EGFR-TKI is administered to the patient in an amount not lower than the standard dose.